1. Introduction
The increasing need to replace animal-based ingredients with plant-based alternatives has gained prominence in aquaculture, driven by a high demand, rising costs, and sustainability challenges associated with animal-derived products [
1]. Plant proteins, such as those from soy and wheat, are widely available, commercially accepted, and considered promising alternatives. However, these sources often contain high levels of dietary fiber, composed of non-starch polysaccharides and lignin, which reduce digestibility [
2].
In this context, diets containing plant-based ingredients, such as soybean meal, are emerging as sustainable alternatives to replace animal-derived components. While soybean meal is widely available and has a favorable amino acid profile, it also contains antinutritional factors, such as trypsin inhibitors and phytic acid, which can negatively impact protein digestibility and intestinal health [
3,
4]. Adverse environmental and nutritional conditions can lead to metabolic disturbances and digestive adaptations [
5,
6], including a reduced feed intake, impaired nutrient digestion, and an increased susceptibility to infections.
Exogenous protease supplementation has shown promise in mitigating the negative effects of antinutritional factors, enhancing digestibility, gut health, and overall fish performance [
7,
8]. Studies have demonstrated that the addition of proteases to soybean-meal-based diets improves protein efficiency, endogenous digestive enzyme activity, and nutrient retention, while also positively impacting the water quality in production systems [
9,
10,
11,
12]. Furthermore, the inclusion of protease reduces diet costs and enhances the economic viability of fish production [
7,
12].
Protease supplementation in fish diets has shown significant benefits for performance, health, and metabolism. Inclusion levels of up to 0.60 g/kg improved the growth and body composition of rohu carp (
Labeo rohita) when poultry by-products were used in the diet [
10]. In European sea bass (
Dicentrarchus labrax), diets containing dried distillers’ grains and exogenous protease reduced the aminotransferase activity levels and promoted growth [
13]. These findings underscore the potential of protease to enhance the growth and health in aquaculture species.
The Nile tilapia (
Oreochromis niloticus) is one of the most consumed fish species worldwide, accounting for 9% of global production and ranking as the third most produced species in 2020 [
14]. In Brazil, tilapia represented 63.93% of the country’s farmed fish production in 2022, marking a 3% increase compared to 2021 [
15]. Its rapid growth, hardiness, and omnivorous feeding habits make it highly favorable for aquaculture systems [
16,
17].
This study aimed to evaluate the effects of diets containing vegetable protein and exogenous protease on the intestinal health and metabolic responses of Nile tilapia.
2. Materials and Methods
2.1. Experimental Diets
A total of six isonitrogenous [36% crude protein (CP)] and isocaloric (18 MJ/kg gross energy) experimental diets were formulated to meet the nutritional requirements of Nile tilapia (O. niloticus). The first diet, soybean meal (SM1), included animal-based protein sources [feather meal, poultry by-product meal, and predominantly fish waste meal (FM)] and plant-based protein sources (corn, wheat bran, and predominantly soybean meal), with an FM:SM ratio of 1:1. The second diet, SM2, had an FM:SM ratio of 1:2, and the third group, SM3, had an FM:SM ratio of 1:3, with ingredient substitutions based on the protein content. Protease was included in the diets at 0 or 0.44 g/kg. This enzyme is a serine protease (EC 3.4.21) and contains 75,000 PROT units/g (supplied by DSM Nutritional Products Ltd., Mszczonow, Maz., Poland). One PROT unit is defined as the amount of enzyme that releases 1 µmol of p-nitroaniline from 1 µM of substrate (Suc-Ala-Ala-Pro-Phe p-nitroaniline) per minute at a pH of 9.0 and a temperature of 37 °C.
The ground ingredients were mixed with oils and distilled water. The mixture was extruded in a single-screw extruder (Inbramaq, model Labor PQ 30, São Paulo, SP, Brazil). The pellets (2.0 mm diameter) were then dried in an oven with forced air recirculation at 55 °C for 24 h. Subsequently, the enzyme was added by spraying with a manual pump, and the diets were stored at −20 °C throughout the experimental period. The composition of the diets and an analysis of the enzyme recovery are presented in
Table 1.
The chemical composition of the experimental diets was determined by following the methods of the Association of Official Analytical Chemists [
19]. Dry matter was determined by oven drying at 105 °C until a constant weight was reached (method 934.01). The mineral matter content was estimated after the incineration of the samples in a muffle furnace at 550 °C for 4 h (method 968.08). The crude protein content (N × 6.25) was determined by the Kjeldahl method after acid digestion (method 954.01). The crude lipid content was determined by the chloroform and methanol extraction method of Bligh and Dyer [
20]. The neutral detergent fiber content of the experimental diets was determined by the method described by Van Soest [
21]. Nitrogen-free extracts (NFE) were calculated according to Bureau et al. [
22].
2.2. Experimental Conditions and Fish-Feeding Management
The experiment was conducted at the Fish Farming Laboratory of UFSM, Campus Palmeira das Missões, RS, Brazil. Nile tilapia were obtained from AquaViva Commercial Fish Farm, Victor Graeff, RS, Brazil. The fish were acclimated to the experimental conditions for two weeks and fed a commercial diet (Supra™, Esteio, RS, Brazil, 36% CP). After acclimation, 360 male juvenile fish (average initial weight: 11.60 ± 0.32 g) were randomly distributed into 18 tanks (usable volume: 220 L), with a stocking density of 20 fish per tank (1 g/L). Each of the six treatments had three replicate tanks. For 49 days, their feed intake was measured daily, and the fish were manually fed until satiety, three times a day (08:00 a.m., 1:30 p.m., and 6:00 p.m.). Siphoning was performed in each tank daily (10:00 a.m. and 4:00 p.m.).
The water temperature and dissolved oxygen were measured daily using YSI ProODO technology (YSI™ Inc., Yellow Springs, OH, USA). Weekly, the total alkalinity (via neutralization titration), total hardness (via complexation titration), pH (YSI™ pH100, Yellow Springs, OH, USA), non-ionized ammonia, and nitrite levels (Alfakit™ colorimetric kit, Florianópolis, SC, Brazil) were evaluated. During the experimental period, the following water quality parameters were recorded: temperature, 25.94 ± 1.00 °C; dissolved oxygen, 5.71 ± 0.57 mg/L; pH, 7.01 ± 0.45; toxic ammonia, 0.03 ± 0.02 mg NH
3/L; nitrite, 0.46 ± 0.09 mg NO
2−/L; alkalinity, 34.62 ± 10.34 mg CaCO
3/L; and hardness, 85.64 ± 4.45 mg CaCO
3/L. All the parameters remained within normal limits for optimal tilapia growth [
3,
16].
2.3. Sample Collection and Analysis
At the end of the experiment, blood samples were collected from nine fish per treatment by puncturing the caudal vein using syringes soaked in heparin. The collected blood was analyzed for erythrocyte parameters. A portion of the blood sample was centrifuged at 3500 rpm at 4 °C for 10 min, and the supernatant was used for a plasma analysis.
The fish were euthanized with an anesthetic overdose [
23] and spinal cord section. Samples of the liver, total muscle, and anterior intestinal tract were collected from nine fish per treatment for biochemical and morphometric analyses.
2.3.1. Intestinal Morphometry
The samples were fixed in a 10% formaldehyde solution for 24 h and subsequently preserved in 70% alcohol. After routine histological processing, the tissues were embedded in paraffin, and blocks were sectioned into 5 μm slices using a microtome (Thermo Scientific™ HM 355S, Walldorf, BW, Germany). Transverse sections were stained with periodic acid-Schiff (PAS) (Sigma-Aldrich™, 3951; Saint-Louis, MO, USA), following the manufacturer’s recommended procedures and adapted from Okuthe and Bhomela [
24]. The histological sections were examined under an Axio Scope A1 microscope (ZEISS™, Oberkochen, BW, Germany), photographed with an Axiocam camera, and analyzed using the ImageJ software (version 1.54d, Bethesda, MD, USA). In the anterior intestine, the height, width, and number of goblet cells in the villi were assessed (54 villi per treatment, totaling 324 villi measured).
2.3.2. Morphometric Indices
Calculated based on the weight and length of the digestive tract, liver, and visceral fat using the following equations: hepatosomatic index (HSI) = [(liver weight/final weight) × 100]; digestive somatic index (DSI) = [(digestive tract weight/final weight) × 100]; celomic fat index (CFI) = [(abdominal cavity fat weight/final weight) × 100]; and intestinal quotient (IQ) = (digestive tract length/total length).
2.3.3. Biochemical Tissue Analysis
Tissue samples (liver and muscle) were heated in a 6 M potassium hydroxide solution at 100 °C for 20 min to analyze the total protein [
25] and glycogen [
26] content. Homogenization in a 20 mM potassium phosphate buffer solution, with a pH of 7.5, followed by centrifugation at 3500 rpm for 10 min, was performed to quantify the amino acids (AAs) [
27] and to assess the activity of the enzymes alanine aminotransferase (ALT) and aspartate aminotransferase (AST) [
28]. For the determination of the total ammonia content [
29], homogenization was performed in a 10% trichloroacetic acid solution.
2.3.4. Plasma Biochemical Analysis
In plasma, the concentrations of total proteins, albumin, and glucose were analyzed using commercial colorimetric kits (Labtest™, Lagoa Santa, Brazil). Serum globulin was calculated by subtracting the albumin values from the total protein. The quantification of amino acids (AAs) was achieved using the methodology described by Spies [
27].
2.3.5. Erythrocyte Parameters
Blood parameters, including the erythrocyte count (RBC), hematocrit (Hct), and hemoglobin (Hb) levels, were measured [
30]. The RBC count was performed after the dilution of 10 µL of blood in a formaldehyde citrate solution, and the count was performed in a Neubauer chamber (Loptik Labor™, Stuttgart, BW, Germany) with the aid of an optical microscope (Bioval™, São Paulo, SP, Brazil). Hct was determined by the microhematocrit technique. The microcapillary was filled with approximately 10 µL (5 cm) of blood and centrifuged at 12,000 rpm for 15 min, after which the cell column was measured using an Hct ruler (Benfer™, Piracicaba, SP, Brazil). The Hb was determined using a commercial colorimetric kit (Labtest™, Lagoa Santa, MG, Brazil), which involved homogenizing 20 µL of blood in a color reagent containing potassium cyanide; subsequently, the absorbance at 540 nm was determined using a spectrophotometer (Bioespectro™, Curitiba, PR, Brazil).
Erythrocyte indices, including the mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), and mean corpuscular hemoglobin concentration (MCHC), were calculated using the following formulas: MCV = (Hct × 10)/RBC; MCH = (Hb × 10)/RBC; and MCHC = (Hb × 100)/Hct [
28].
2.4. Statistical Analysis
All the data were analyzed using the R™ software, version 4.3.0 (R Foundation for Statistical Computing, Vienna, Austria), and graphs were created using the SigmaPlot™ software, version 14.5 (Systat Software Inc., San Jose, CA, USA). The data were subjected to a Shapiro–Wilk normality analysis. Two-way ANOVA was used to analyze the individual effects of diets and exogenous protease and the interaction among them. The Tukey test was applied if there was a significant difference (p < 0.05), and the results are presented as the means and standard error (±SE).
